Rapid prediction of G protein-coupled receptor structures using nanoluciferase assay / 药学学报

Using beta-2 adrenergic receptor, 5-hydroxytryptamine and angiotensin II type 1 receptor as control, we here established a method for rapid prediction of the initial position amino acids of N-terminal, C-terminal, intracellular loops, extracellular loops and transmembrane (TM) regions in G protein-coupled receptors (GPCRs), and successfully predicted the structure of Mas-related G protein-coupled receptors X3 (MRGPRX3). To achieve this purpose, nanoluciferase (Nluc) was inserted into the different sites of these GPCRs′ sequence by sequence and ligation-independent cloning (SLIC) method, and the luminescence value were measured to distinguish the different parts of GPCRs. The results showed that luminescence values of NLuc luciferase at TM region were less than 100 000, and the values were higher than 1 000 000 at N terminal, C terminal, or extracellular loops and intracellular loops, and the values were between 100 000 and 500 000 at junction. The predicted MRGPRX3 structure was analyzed in detail and was compared with AlphaFold predicted structure. In conclusion, this method could provide useful information of GPCR structure model for the ligand virtual screening, and could provide certain experimental basis for structural pharmacology.

Adaption and Optimization of Universal Hairpin Transduction in Gene Diagnostics Based on Nucleic Acid Circuits / 分析化学

Isothermal nucleic acid amplifications, as powerful as polymerase chain reaction but functioning at a constant temperature, are considered to be very promising technique in achieving point-of-care gene diagnostics. However, until now, their practical applications are still seriously lagged by the bad reliability resulting from the problems such as false positive amplification and low signal amplitude. In this work, a universal transduction method in which any sequence ( including loop-mediated isothermal amplification products) could be transduced via a hairpin transducer into a catalyst of a well-engineered circuit (catalytic hairpin assembly, CHA) was established. Because CHA circuit could amplify tens to hundreds fold with especially high sequence specificity, it could provide both accuracy and high amplitude for sequence detection. And for a new targeting sequence, the only sequence needed to be changed was the hairpin transducer. Due to the importance of the transducer, we provided and verified a universal designing rule-set to guarantee the transducing efficiency ( signal to background ratio) of the transducer. Transducers designed following this rule set were then proved to be very efficient in detecting pathogen gene targets. As less as near single molecule ( 20 copies ) of pathogen genes could be detected with significant fluorescent and electrochemical signals.

Frequency Distribution Features of Innate-like Lymphocytes in Peripheral Blood of Normal Adults / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the frequency distribution features of innate-like lymphocytes (iNKT cells, γΔT cells and B1 cells) in peripheral blood of normal adults.</p><p><b>METHODS</b>The flow cytometry with 6 fluorescence staining was used to detect the percentages of iNKT lymphocytes, γΔT lymphocytes, B1 lymphocytes and adaptive T lymphocyte, B2 lymphocytes in peripheral blood lymphocytes of 50 normal adults. The difference and correlation between these lymphocyte subsets were analyzed by statistical software.</p><p><b>RESULTS</b>The percentage of iNKT cells in peripheral blood of 50 normal adults was 0.18% (0.01%-2.01%), the percentage of γΔT cells was 4.90% (1.45%-20.14%), the percentage of B1 lymphocytes was 1.62% (0.20%-3.77%), the percentage of adaptive T cells was 63.52% (33.20%-83.22%), the percentage of B2 cells was 6.64% (3.07%-13.80%). B1 and B2 were two subsets of B lymphocyte, the percentage of B2 in B lymphocyte was 81.43% (57.90%-94.12%) and more than that of B1 lymphocyte; the percentage of B1 lymphocytes was 17.28% (5.28%-41.13%). In T lymphocyte group the percentage of iNKT cell was 0.32% (0.01%-3.6%), the percentages of γΔT cells and adaptive T cells were 7.55% (3.04%-27.66%) and 91.98% (72.22%-96.86%) respectively. Spearman correlation analysis was used to analyze the correlation between the percentages of several lymphocyte subsets. There was a positive correlation between iNK T cells and γΔT cells, γΔT cells and adaptive T cells, B1 cells and B2 cells (r=0.39, P=0.0056; r=0.6028, P<0.0001; r=0.4791, P=0.0004). It was also found that the percentage of iNKT cells in female peripheral blood lymphocytes was 0.29% (0.06%-2.01%), and significantly higher than that in male peripheral blood lymphocytes 0.12% (0.01%-1.37%) (P<0.05).</p><p><b>CONCLUSION</b>The percentages of γΔT cells, B1 cells and iNKT cells in peripheral blood lymphocytes of normal adults are significantly lower than that of adaptive lymphocytes, and their contents in peripheral blood decrease in turn. There are no sex differences in the percentages of these lymphocyte subsets except iNKT cells.</p>